Nature contains many microbial species that have never been cultivated. In the past decade researchers have identified microorganisms in nature by direct molecular techniques. These ideas were first formulated by Pace and co-workers, who proposed that natural ecosystems harbored microbial species that were unknown to scientists (Olsen et al., Ann. Rev. Microbiol. 40:337-366, 1986; Pace, et al., Cell 45:325-326, 1986; Woese, Microbiol. Rev. 51:221-271, 1987). By cloning 16S rRNA genes directly from natural ecosystems, it has been possible to catalog a remarkable spectrum of species of unique phylogenetic descent, but for which no laboratory cultures exist (Giovannoni, et al., Nature 345:60-63, 1990; Kruske et al. Appl. Environ. Microbiol. 63:3614-3621, 1997; Pace, Science. 276:734-740, 1997).
The introduction of genetic methods for studying microbial diversity was prompted by the observation that, in many ecosystems, fewer than one cell in one thousand produces a colony on a Petri dish containing nutrient agar—a standard laboratory method of growing heterotrophic bacteria. These observations were dubbed “the great plate count anomaly” (Staley and Konopka, Ann. Rev. Microbiol. 39:321-346, 1985). One possible simple explanation for these results is that natural ecosystems harbor microbial species that can not be grown by the standard methods often used by microbiologists.
Techniques for cloning 16S rRNA genes directly from DNA isolated from natural ecosystems circumvented the problem of having to grow microbes to identify them. With this approach it has been possible to catalog from nature a remarkable diversity of prokaryotic species that often represent unique phylogenetic lines of descent, but for which no cultures exist (Barns et al., Proc. Natl. Acad. Sci USA. 91:1609-1613, 1994; DeLong Proc. Natl. Acad. Sci. 89:5685-5689, 1992; Mullins et al., Limnol. Oceanogr. 40:148-158, 1995). These results might well be regarded as a revolution.
Many scientific fields—environmental microbiology, medicine, enzymology, exobiology and genomics—have been impacted by the past decade of discovery of novel, uncultured microbial diversity in the natural world (Pace, Science. 276:734-740, 1997; Robertson et al., SIM News 46:3-8, 1996).
Molecular studies of in situ microbial diversity are entering a phase of synthesis. Early studies invariably uncovered exciting evidence that ecosystems such as seawater, soil, hot springs and fresh water were populated with novel organisms, prompting numerous laboratories to adopt and apply similar methods. Microbial diversity was viewed by many as a terra incognita, and the spoils were rich new additions to the 16S rRNA gene tree depicting evolutionary themes among cells (Barns et al., Proc. Natl. Acad. Sci. USA. 91:1609-1613, 1994). This early phase of discovery led to a phase of synthesis, with the recognition that many of the major unknown gene lineages are widely distributed and therefore are encountered repeatedly in clone libraries prepared by different investigators (Kruske et al., Appl. Environ. Microbiol. 63:3614-3621, 1997; Methe et al., Limnol. Oceanog. 43:368-374, 1998; Mullins et al., Limnol. Oceanogr. 40:148-158, 1995).
Seawater, one of the first ecosystems to be studied by gene cloning, and now the best known, serves as an example to illustrate the very great significance of a few key lineages of organisms. Of 616 bacterial 16S rRNA genes recovered from seawater samples from seven sites, 80% fall into only nine phylogenetic groups. Cultured species are known for only two of these groups (Giovannoni et al., pp. 63-85, In Evolution of microbial life, Roberts et al., eds., Cambridge University Press, Cambridge, 1996). This analysis does not include the Group I and Group II marine archaea, because generally their genes are cloned by different methods (DeLong, Proc. Natl. Acad. Sci. 89:5685-5689, 1992). Both of these archaeal groups are also uncultured, and their addition raises the number of really abundant marine bacterioplankton groups to ten. Moreover, two of the unique gene lineages first found in seawater were later found also to be abundant in freshwater: the SAR11 lineage of the proteobacteria, and the Group I crenarchaeota.
Similar conclusions are now emerging from studies of freshwater and soils. In one of the best examples, Kruske et al. analyzed soil microbiota from the arid Southwestern United States (Appl. Environ. Microbiol. 63:3614-3621, 1997). They observed that five novel clusters of organisms, all having no cultured representatives, accounted for 64% of the genes recovered. More significantly, a thorough survey of data from other studies led to the conclusion that these unknown organisms were widely distributed geographically. Studies of freshwater systems support similar conclusions: lineages such as the SAR11 group, the Acidobacterium group, the Verrucomicrobium group, and the freshwater actinomycete clade, are found in nearly every example examined (Bel'kova et al., Doklady Biol. Sci. 348:692-695, 1996; Hioms et al., Appl. Environ. Microbiol. 63:2957-2960, 1997; Methe et al., Limnol. Oceanog. 43:368-374, 1998; Wise et al., Appl. Environ. Microbiol. 63:1505-1514, 1997).
The experimental approach of growing microorganisms from natural systems by dilution of a natural inoculum into media with very low dissolved organic carbon (DOC) concentrations has been described previously, and is considered in a recent review by Schut and colleagues (Schut et al., Aquat. Microb. Ecol. 12:177-202, 1997). Despite the success of this approach, it has never been applied to large numbers of cell cultures in small volumes.
There is still a strongly felt need for methods of culturing and identifying novel microorganisms, and for identifying and analyzing positive and negative interactions between microorganisms in culture. It is to these needs that the current invention is addressed.